Current evidence suggests that the optimal vaccines for cancer should incorporate tumor-specific cytotoxic as well as helper epitopes. Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines, which could combine multiple tumor epitopes defined by CD8 (+) cytotoxic T lymphocytes (CTL), as well as CD4 (+) T-helper (Th) cells. Whereas several HLA-A*0201-restricted CTL-defined wt p53 peptides are available for vaccine use, no Th-defined wt p53 peptides are known. In order to identify such peptides, we generated anti-p53 CD4 (+) T cells from peripheral blood mononuclear cells (PBMC) obtained from an HLA-DRB1*0401(+) donor by in vitro stimulation with dendritic cells pulsed with recombinant human p53 protein. A panel of eight algorithm-predicted HLA-DR4-binding wt p53 peptides was tested for recognition by the anti-p53 Th cells. The anti-p53 Th cells identified the wt p53 (110-124) peptide as a naturally presented epitope. Subsequent in vitro-based experiments demonstrated the ability of the anti-wt p53 (110-124) Th cells to enhance the generation and anti-tumor functions of CD8 (+) effector cells and its utility as a vaccine component. Although HLA-DR4 is the most commonly expressed class II MHC allele in humans, it is still only expressed in 20% of individuals, which limits the applicability for the wt p53 110-124 peptide. We, therefore, initiated a research aimed at identifying a "pan HLA-DR" binding wt p53 peptide. Using a protocol similar to the one detailed above, we have identified the wt p53 25-35 peptide as capable of inducing anti-p53 Th cells from PBMC obtained from 6/6 HLA-DR donors. These findings strongly suggest that this peptide might well be a suitable wt p53 helper peptide for even more broadly applicable p53-based vaccine that employs cytotoxic as well as helper peptides than the one employing the wt p53 110-124 helper peptide. The T cell receptor (TCR) from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D (b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been expressed in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicated that human p1049/A2 had a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D (b). However, T cell binding and cytolytic activity were independent of CD8 when stimulated with human p1049/A2. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produced p56 (lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrated that CD8 is recruited to the site of TCR:peptide MHC binding. These results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement. Recently, we have determined the co-crystal structure of our xenoreactive T cell receptor, AHIII 12.2, in complex with its non-self MHC ligand, A2-p1049. This AHIII 12.2 TCR docks onto the A2-p1049 surface in a manner unlike any other TCR/MHC complex for which a co-crystal structure has been determined. The orientation of this xenoreactive murine TCR atop human MHC deviates from the typical orientation more than any previously determined TCR/MHC structure. The shift in TCR topography in the A2-p1049/AHIII structure is almost exclusively due to a displacement of the AHIII TCR Vb domain. This Vb domain displacement is seen in some of the other TCR/MHC co-crystal structures, however, we are the first recognize this alteration. Interestingly, a TCR Vb domain shift completely correlates with CD8 co-receptor independence in all known TCR/MHC structures. Therefore, we have proposed that the orientation seen in the TCR recognition of MHC is a consequence of selection during T cell development and to understand the molecular basis for this unique structural finding we are producing an AHIII 12.2 TCR transgenic mouse.